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120 CHAPITRE 3. STRUCTURE DE L’HC DE C. MAENAS PAR ESI-MS FIG. 3.5 – Acidic reassociation of Carcinus maenas hemocyanin by formic acid. Left part, mass spectra of a whole hemolymph sample dissociated in 10 mM AcNH4, 0.03 % TEA and acidified with increasing quantities of formic acid (0, 0.004, 0.0045 and 0.04 % formic acid for pH 8.55, 7.94, 7.7 and 4.22, respectively). For pH 4.22, the isoelectric point is passed since a precipitate forms for slightly higher pH values. m, monomers peaks, h, hexamer peaks (450 kDa), d, dodecamer peaks (900 kDa), dl, light dodecamer peaks (885 kDa). Right part, focus for each mass spectrum on the 4300-4600 m/z range, in which the 17 H+ charged peak of each monomer is expected. The presence or absence of each dissociated subunit can be determined by examining the peaks visible in this m/z range.
3.2. MANUSCRIT : STRUCTURAL STUDY OF C. MAENAS HC BY ESI-MS 121 Reassociation by L-lactic acid L-lactic acid also induced a partial reassociation of the subunits into complexes, as observed with formic acid (figure 3.6). The main complex observed is the light dodecamer, with a relative intensity higher that in the case of formic acid reassociation. A preferential mobilization of the lightest subunits is observed again, which is consistent with the appearance of light dodecamer. While this preferential recruitment is also observed with formic acid, there is an important difference since L-lactic acid induces an immediate disappearance of the two light subunits (Cm1 and Cm2) as soon as it is added, even if the pH is maintained over 8.4. This specific and immediate effect contrasts with the progressive effect observed with formic acid. Cm1 and Cm2 remain undetectable at pH 5.9, over the isoelectric point. Experiments on purified complexes (dodecamers and hexamers) Dissociation/reassociation experiments were performed on purified complexes, using formic acid as the acidifying agent. Figure 3.7 presents the results obtained for purified dodecamers and hexamers. In both cases, dissociation is induced at alkaline pH. When pH is returned to 7.5, dissociated dodecamer subunits reassociate partially into dodecamers and light dodecamers (a very small signal for hexamers is also visible) whereas dissociated hexamer subunits only reassociate up to hexamers and no dodecamers is formed. Hexamers were not fully dissociated upon alkalinization ; however for dodecamers also the efficiency of the dissociation could vary from one experiment to another. Chelation of divalent cations by EDTA When divalent cations are removed by EDTA washing after alkaline dissociation of whole hemolymph preparation, returning to pH 7.52 by washing with a 10 mM AcNH4, 0.005 % TEA solution induces a partial reassociation mainly into hexamers and to a lesser extent into dodecamers (figure 3.8). Interestingly, all dissociated monomers peaks disappear upon reassociation except for the 4450 peak corresponding to the subunit specific of the dodecameric assembly. Addition of L-lactic acid to the sample after removal of EDTA still resulted in the disappearance of the lightest subunits peaks (figure 3.8). No major dissociation into hexamers or monomers was observed upon addition of EDTA at neutral pH (data not shown).
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THESE DE DOCTORAT DE L’UNIVERSITE
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i Résumé / Abstract Réponse adap
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iv SOMMAIRE 4.2 Objectifs de l’é
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2 CHAPITRE 1. INTRODUCTION Les éco
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4 CHAPITRE 1. INTRODUCTION 1.1 Mili
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6 CHAPITRE 1. INTRODUCTION Les prin
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8 CHAPITRE 1. INTRODUCTION TAB. 1.1
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10 CHAPITRE 1. INTRODUCTION et cycl
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FIG. 1.5 - Localisation des princip
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(a) FIG. 1.6 - Fonctionnement d’u
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16 CHAPITRE 1. INTRODUCTION TAB. 1.
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18 CHAPITRE 1. INTRODUCTION FIG. 1.
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20 CHAPITRE 1. INTRODUCTION Différ
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24 CHAPITRE 1. INTRODUCTION le mili
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26 CHAPITRE 1. INTRODUCTION Capacit
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28 CHAPITRE 1. INTRODUCTION TAB. 1.
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30 CHAPITRE 1. INTRODUCTION 700 600
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32 CHAPITRE 1. INTRODUCTION férent
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34 CHAPITRE 1. INTRODUCTION (a) (b)
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40 CHAPITRE 1. INTRODUCTION 1 P 50
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42 CHAPITRE 1. INTRODUCTION presque
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44 CHAPITRE 1. INTRODUCTION affecte
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50 CHAPITRE 1. INTRODUCTION fure co
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52 CHAPITRE 1. INTRODUCTION amélio
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54 CHAPITRE 1. INTRODUCTION 1.5 Que
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56 CHAPITRE 2. ESI-MS ET MALLS APPL
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170 CHAPITRE 4. PLASTICITÉ PHÉNOT
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172 CHAPITRE 5. ADAPTATIONS RESPIRA
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216 CHAPITRE 6. CONCLUSION ET PERSP
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Annexe A Détermination des masses
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229 10000 h280 .M h) h280−h337 80
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Annexe B Détermination des distrib
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233 Modèle 2 : 2 sous-unités lég
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235 1 0.9 0.8 6 s-u aléatoires 2 s
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Bibliographie Adair, G. S. The hemo
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BIBLIOGRAPHIE 239 Catalina, M. I.,
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BIBLIOGRAPHIE 241 Farmer, C. S., J.
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BIBLIOGRAPHIE 243 Hourdez, S. Adapt
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BIBLIOGRAPHIE 245 Mangum, C. P. Sub
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BIBLIOGRAPHIE 247 Rainer, J., C. P.
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BIBLIOGRAPHIE 249 Svedberg, T. et I
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BIBLIOGRAPHIE 251 Vanin, S., E. Neg
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Liste des figures 1.1 Cycle des mar
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LISTE DES FIGURES 255 4.17 Profils
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Table des matières Résumé Sommai
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TABLE DES MATIÈRES 261 4.3.5 Spect
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TABLE DES MATIÈRES 263 Table des m
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